Intracellular pathogens pose a unique challenge to host immunity and medical intervention, making this class of pathogens one of the largest contemporary human health burdens. Listeria monocytogenes, a gram- positive, intracellular pathogen, activates two distinct innate immune pathways, delineated by the intracellular location (phagasomal or cytosolic) of infecting bacteria. A unique response to cytosolic bacteria has led to the hypothesis of a cytosolic surveillance pathway (CSP) capable of identifying pathogenic microbes that compromise the cytosol. Activation of this pathway leads to IFN-2 production and has been demonstrated with multiple intracellular pathogens, including M. tuberculosis, F. tularensis, L. pneumophila, Brucella, and T. cruzi. Detection of live, replicating intracellular microbes is dependent on host receptors that bind microbial ligands. Currently, we lack an understanding of the host-receptors and microbial-ligands involved in innate immune detection of L. monocytogenes, as well as many other pathogens of this class. Detailed analysis of CSP activation by L. monocytogenes showed that IFN-2 production is almost entirely dependent on the presence the multi-drug transporter MdrM. Based on this we hypothesized the presence of a bacterial derived small molecule able to activate the CSP. Now, we have purified and identified a novel ligand derived from L. monocytogenes that can directly activate the CSP. Bacteria that are unable to generate this ligand will be used to characterize its role in pathogenesis in both cell culture and murine models of infection. Furthermore, use of various host strains will allow for the characterization of the innate immune pathways activated by this ligand during infection. Finally, we will use immobilized ligand to identify interacting host proteins with the aim of determining the host receptor. Cell culture models will be used to validate identified ligand interacting proteins. PUBLIC HEALTH RELEVANCE: Given the important role of innate immunity in human disease and the potent response elicited by the cytokines generated from innate immune activation, a detailed understanding of the mechanism by which L. monocytogenes activates the immune system may give rise to many promising therapeutics. Immuno- modulating molecules from L. monocytogenes and the host proteins that recognize these molecules will provide targets that can be manipulated to treat various forms of human disease, with the most obvious applications toward new adjuvants, vaccines to treat microbial infection and malignancies, and novel interferon inducing therapies for cancer and viral treatment.